Compositions and related methods for modulating alkaloid production by controlling PMT promoter activation mediated by transcriptional factors ERF and MYC

ABSTRACT

Compositions and methods for modifying the production levels of alkaloids in plants are provided. Alkaloid production can be genetically controlled by modulating the transcriptional activation of PMT genes mediated by members of the ERF family and/or Myc family of transcription factors. Novel nucleotide sequences encoding the Myc family of transcription factors are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 12/676,871, filed on Nov. 10, 2010, which is a U.S.National Stage pursuant to 35 U.S.C. §371 of International PatentApplication PCT/US2008/010447, filed on Sep. 5, 2008, and published asWO 2009/035547 on Mar. 19, 2009, which claims priority to U.S.Provisional Patent Application No. 60/935,948, filed on Sep. 7, 2007,and U.S. Provisional Patent Application No. 60/935,947, filed on Sep. 7,2007, all of which are incorporated herein by reference in theirentireties for all purposes.

Incorporated by reference in its entirety herein is a computer-readablesequence listing submitted concurrently herewith and identified asfollows: One (34,561 Byte ASCII (Text)) file named “14311684 sequencelisting.txt,” created on Jan. 10, 2017.

BACKGROUND

Nicotine production from polyamine putrescine, a precursor of nicotine,can be produced by two pathways in plants. Putrescine can be synthesizeddirectly from ornithine in a reaction catalyzed by the enzyme ornithinedecarboxylase, or can be produced indirectly from arginine in a sequenceof reactions initiated by arginine decarboxylase. The first committedstep in nicotine biosynthesis is the conversion of putrescine toN-methyl putrescine by putrescine N-methyltransferase (“PMT”).N-methylputrescine is subsequently oxidized by a diamine oxidase, and iscyclized to produce a 1-methyl-Δ¹-pyrrolium cation, which issubsequently condensed with nicotinic acid to produce nicotine.

There is a need for compositions and improved methods for geneticallyregulating the production levels of nicotine and other alkaloids inplants, including transgenic plants, transgenic tobacco plants,recombinant stable cell lines, recombinant stable tobacco cell lines,and derivatives thereof.

SUMMARY

In various embodiments, compositions and methods for modifying theproduction levels of nicotine and other alkaloids in plants areprovided. Nicotine and other alkaloid production can be geneticallymodified by modulating the transcriptional activation of PMT genesmediated by members of the ERF family and/or Myc family oftranscriptional factors.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates multiple signal transduction pathways, induced byvarious stimuli that regulate PMT promoter activation/repression inplants.

FIGS. 2A-2C show the identification of transcription factors of the ERFfamily that can bind specifically to the GAG motif.

FIGS. 3A-3C show relative expression levels of transcripts for NtERF2,NtERF3, and NtERF5, measured by RT-PCR in BY-2 cell extracts. Theexpression levels of NtERF2 and NtERF5 were responsive to MeJA-induciblepathway. The expression level of NtERF3 was responsive to ethylene (orethephon) inducible pathway.

FIG. 4 shows that NtERF5 can bind to the GAG motif by in vitro gelmobility shift assay.

FIGS. 5A-5C show that NtERF5 can activate the NtPMT1a promoter bybinding to the AT-rich region of the GAG motif.

FIG. 6A shows that NtERF5 over-expression results in GUS reporterexpression in a MeJA-dependent manner by binding to 4xGAG motifspositioned upstream of a promoter that drives GUS reporter expression.Overexpressed cell lines (“OE”) that have been stably integrated with4XGAG::GUS reporter were transiently transfected with expression vectorsover-expressing NtERF5.

FIG. 6B shows that NtERF5 over-expression is not sufficient to activateNtPMT expression in the absence of MeJA. NtPMT transcripts were detectedin extracts of cell lines that were induced by MeJA.

FIG. 7 shows that NtERF2 over-expression activates NtPMT expression in aMeJA-independent manner.

FIGS. 8A-8C show that Myc3/Myc4 over-expression in BY-2 cells isinducible by MeJA.

FIG. 8D shows that Myc3/Myc4 over-expression in the roots of transgenictobacco is inducible by MeJA.

FIG. 9 shows that Myc3 binds the G-box of the GAG motif by in vitromobility shift assay.

FIG. 10 shows differential localization of Myc3 and Myc4, respectively,in BY-2 cells after MeJA exposure. NtMyc3-GFP localized to the nucleusafter MeJA exposure. In contrast, NtMyc4-GFP was excluded from thenucleus after MeJA exposure.

FIG. 11 shows the polynucleotide sequence for NtMyc3 cDNA (SEQ ID NO:1).

FIG. 12 shows the polypeptide sequence for NtMyc3 protein (SEQ ID NO:2).

FIG. 13 shows the polynucleotide sequence for NtMyc4 cDNA (SEQ ID NO:3).

FIG. 14 shows the polypeptide sequence for NtMyc4 protein (SEQ ID NO:4).

FIG. 15 shows the polynucleotide sequence for NtERF2 cDNA (SEQ ID NO:5).

FIG. 16 shows the polynucleotide sequence for NtERF5 cDNA (SEQ ID NO:6).

DETAILED DESCRIPTION

Regulation of nicotine biosynthesis is desirable in a variety of plants,especially in tobacco plants. Nicotine biosynthesis can be regulated bycontrolling the expression levels and/or the activities of enzymesinvolved in the nicotine biosynthetic pathway. In particular, aneffective way to genetically regulate nicotine production is bycontrolling the transcriptional activation of promoters that control theexpression of genes encoding putrescine N-methyltransferases (“PMT”).PMT is one of several critical enzymes involved in the nicotinebiosynthetic pathway in plants, including tobacco plants. Variouscompositions and methods for modifying PMT expression levels in plantsare provided, as further described below.

The production levels of nicotine, other alkaloids, and secondarymetabolites can be genetically regulated by controlling the expressionlevel of PMT, which correlates directly with their production levels.This can be accomplished by controlling PMT promoter activation thatcorrelates with the expression levels of the PMT structural gene. PMTRNA transcripts produced by transcriptional processes are subsequentlytranslated into PMT polypeptides that exhibit PMT enzymatic activity.The activation of the PMT promoter by sequence-specific transcriptionalfactors (“transcriptional activators”) can increase the levels of PMTRNA transcripts and PMT polypeptides produced. In contrast, therepression of the PMT promoter by sequence-specific transcriptionalfactors (“transcriptional repressors”) can decrease the levels of PMTRNA transcripts and PMT polypeptides produced.

PMT promoter activation is responsive to various endogenous andexogenous signals, including phytohormones, wounding, and invasion bypathogens or insects. FIG. 1 illustrates the existence of multiplesignal transduction pathways inducible by various phytohormones,including jasmonic acid (“JA”), auxin, and ethylene, which can affectPMT promoter activation. As shown in FIG. 1, the transcriptionalregulation of PMT genes can be responsive to multiple signaltransduction pathways that can be co-induced if multiple stimulantsexist simultaneously in an environment. When a particular signaltransduction pathway is induced, the expression level and/or thetranscriptional activity of a transcriptional factor can be increased.Alternatively, the expression level and/or the transcriptional activityof a transcriptional factor can be decreased by inducing a particularsignal transduction pathway. For example, a JA-inducible signaltransduction pathway can be blocked by co-exposure to sufficientconcentrations of auxin and/or ethylene that appear to have antagonisticeffects on the JA-inducible pathway. In particular, PMT promoteractivation induced by JA exposure can be blocked by exposure to auxinand/or ethylene.

In various plants, such as tobacco, the phytohormone jasmonic acid(“JA”) (3-oxo-2-(cis-2-pentenyl)-1-cyclopentaneacetic acid) and/or itsmethylated ester derivative (“MeJA”) can be utilized for inducingnicotine production. JA and/or MeJA can be utilized for inducing theexpression of genes involved in the biosynthesis of secondarymetabolites such as alkaloids, flavonoids, and terpenoids. Furthermore,JA and/or MeJA can be utilized to control various processes thatregulate plant growth and environmental adaptation, including seedgermination, regulation of carbon and nitrogen storage, photosynthesis,senescence, pollen development, fruit ripening, wound responses, andresistance to insects and pathogens. For regulating defense responses,JA and/or MeJA can be utilized to act synergistically with, orantagonistically against, two other plant hormones, salicylic acid (SA)and ethylene. JA and/or MeJA can be utilized for inducing the expressionof genes encoding proteinase inhibitors involved in pest resistance andgenes encoding defensins that exhibit antimicrobial activity.

Therefore, various wound-induced and stress-induced biological responsesof interest can be elicited by controlling the amount and extent ofexposure to such phytohormones.

In Nicotiana tabacum, at least five PMT genes have been characterized:NtPMT1a, NtPMT1b, NtPMT2, NtPMT3, and NtPMT4. PMT gene expression in theroots of N. tabacum can be up-regulated by various stimuli, includingtopping procedures, the physical invasion by insects and/or otherherbivores, and JA or MeJA exposure.

This disclosure describes a regulatory region upstream of the corepromoter elements of PMT promoters, referred to as the “GAG motif,” thatconfers PMT promoters with responsiveness to JA and MeJA. The GAG motifcomprises a G-box like element, an AT-rich element, and a GCC-like boxelement. The GAG motif functions optimally as an intact, tripartiteunit, in that the three elements must be utilized together, and must bearranged so that the G-box like element is positioned upstream of theAT-rich element, which is positioned upstream of the GCC-like boxelement.

Based on experimental studies described herein, the GAG and the G-Gderivative fragment (similar to the structure of the GAG motif butdeficient in the AT-rich element) have been shown to be responsive to JAand MeJA when plants are exposed to these phytohormones. The JA and

MeJA responsiveness of the GAG motif can be mediated by members of theERF and Myc families of transcriptional factors, as described herein.The GAG motif confers the recruitment of ERF and Myc transcriptionalfactors and other transcriptional factors when operably-linked to apromoter of interest, which is operably-linked to a transgene ofinterest positioned downstream of the promoter of interest. Suitabletransgenes include genes that encode various enzymes involved in thebiosynthesis of alkaloids, nicotine, and flavonoids, for example.

The sub-elements of the GAG motif that can recruit particular members ofthe ERF and Myc families have been further characterized by resultsdescribed herein. For example, the experimental results show thatERF2/ERF3/ERF5 are recruited to the GAG motif in a JA-inducible manner.This occurs through the GCC-like box element and requires some or all ofthe AT-rich element for recruitment. For example, the experimentalresults show that the G-box element can recruit NtMyc3/NtMyc4 in aJA-inducible manner.

For up-regulating or down-regulating various promoters of interest, thefollowing compositions and methods are contemplated:

In general, various embodiments are directed to expression vectors thatenable the over-expression of transcriptional factors, NtMyc3, NtMyc4,NtERF2, and/or NtERF5, for modulating the production levels of nicotine,other alkaloids, including various flavonoids. These expression vectorscan be transiently introduced into host plant cells or stably integratedinto the genomes of host plant cells to generate transgenic plants byvarious methods known to persons skilled in the art. When theseexpression vectors are stably integrated into the genomes of host plantcells to generate stable cell lines or transgenic plants, theover-expression of transcriptional factors, NtMyc3, NtMyc4, NtERF2,and/or NtERF5, can be utilized as a method for modulating the promoteractivation of endogenous promoters that are responsive to thesetranscriptional factors. Furthermore, such host plant cells can befurther manipulated to receive heterologous promoter constructs that areresponsive to transcriptional factors, NtMyc3, NtMyc4, NtERF2, and/orNtERF5. Furthermore, such host plant cells can be further manipulated toreceive heterologous promoter constructs that have been modified byincorporating one or more GAG motifs upstream of the core elements ofthe heterologous promoter of interest.

Any promoter of interest can be manipulated to be responsive to JA andMeJA by incorporating one or more GAG motifs and/or derivative GAGmotifs upstream of the promoter of interest. Suitable promoters includevarious promoters of any origin that can be activated by thetranscriptional machinery of plant cells, such as various homologous orheterologous plant promoters and various promoters derived from plantpathogens, including bacteria and viruses. Suitable promoters includeconstitutively active promoters and inducible promoters.

For various expression vectors described below, various genes thatencode enzymes involved in biosynthetic pathways for the production ofalkaloids, flavonoids, and nicotine can be suitable as transgenes thatcan be operably-linked to a promoter of interest.

In another embodiment, an expression vector comprises a promoteroperably-linked to the cDNA encoding Myc3, Myc4, ERF2, and/or ERF5. Inanother embodiment, a plant cell line comprises an expression vectorcomprising a promoter operably-linked to the cDNA encoding Myc3, Myc4,ERF2, and/or ERF5. In another embodiment, a transgenic plant comprisesan expression vector comprising a promoter operably-linked to the cDNAencoding Myc3, Myc4, ERF2, and/or ERF5. In another embodiment, methodsfor genetically modulating the production of alkaloids, flavonoids, andnicotine are provided, comprising: introducing an expression vectorcomprising a promoter operably-linked to the cDNA encoding Myc3, Myc4,ERF2, and/or ERF5.

In another embodiment, an expression vector comprises a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved inthe biosynthesis of alkaloids. In another embodiment, a plant cell linecomprises an expression vector comprising a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved inthe biosynthesis of alkaloids. In another embodiment, a transgenic plantcomprises an expression vector comprising a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved inthe biosynthesis of alkaloids. In another embodiment, methods forgenetically modulating the production level of alkaloids are provided,comprising: introducing an expression vector comprising a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved inthe biosynthesis of alkaloids.

In another embodiment, an expression vector comprises a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved inthe biosynthesis of flavonoids. In another embodiment, a plant cell linecomprises an expression vector comprising a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved inthe biosynthesis of flavonoids. In another embodiment, a transgenicplant comprises an expression vector comprising a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved inthe biosynthesis of flavonoids. In another embodiment, methods formodulating the production level of flavonoids are provided, comprising:introducing an expression vector comprising a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved inthe biosynthesis of flavonoids.

In another embodiment, an expression vector comprises a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved innicotine biosynthesis. In another embodiment, a plant cell linecomprises an expression vector comprising a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved innicotine biosynthesis. In another embodiment, a transgenic plantcomprises an expression vector comprising a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved innicotine biosynthesis. In a preferred embodiment, the enzyme is PMTinvolved in nicotine biosynthesis. In another embodiment, methods forgenetically modulating the production level of nicotine are provided,comprising: introducing an expression vector comprising a first promoteroperably-linked to cDNA encoding Myc3, Myc4, ERF2, and/or ERF5; and asecond promoter operably-linked to cDNA encoding an enzyme involved innicotine biosynthesis. FIG. 11 shows the polynucleotide sequence forNtMyc3 cDNA (SEQ ID NO:1). FIG. 12 shows the polypeptide sequence forNtMyc3 protein (SEQ ID NO:2). FIG. 13 shows the polynucleotide sequencefor NtMyc4 cDNA (SEQ ID NO:3). FIG. 14 shows the polypeptide sequencefor NtMyc4 protein (SEQ ID NO:4). FIG. 15 shows the polynucleotidesequence for NtERF2 cDNA (SEQ ID NO:5), which encodes forEthylene-responsive transcription factor 2 (Swiss Protein Accession No.Q40479; Plant Cell 7 (2), p 173-182 (1995; PUBMED #7756828). FIG. 16shows the polynucleotide sequence for NtERF5 cDNA (SEQ ID NO:6), whichencodes ERF Transcription Factor 5 (NCBI Accession No. AY655738; Mol.Plant Microbe Interact. 17(10), p 1162-1171 (2004); PUBMED #15497409).NtERF5 cDNA (SEQ ID NO:6) also refers to sequence published in Mol.Plant Microbe Interact. 17(10), p 1162-1171 (2004).

Another embodiment is directed to an isolated cDNA encoding NtMyc3 (SEQID NO:1), or fragments thereof. Another embodiment is directed to anisolated cDNA encoding NtMyc3 and having at least 85%, 90%, 95%, 96%,97%, 98%, and 99% sequence identity to SEQ ID NO:1, or variant fragmentsthereof.

Another embodiment is directed to an isolated cDNA encoding NtMyc4 (SEQID NO:3), or fragments thereof. Another embodiment is directed to anisolated cDNA encoding NtMyc4 and having at least 85%, 90%, 95%, 96%,97%, 98%, and 99% sequence identity to SEQ ID NO:3, or variant fragmentsthereof.

Another embodiment is directed to an isolated polypeptide NtMyc3 (SEQ IDNO:2), or fragments thereof. Another embodiment is directed to anisolated polypeptide NtMyc3 and having at least 85%, 90%, 95%, 96%, 97%,98%, and 99% sequence identity to SEQ ID NO:2, or fragments thereof.

Another embodiment is directed to an isolated polypeptide NtMyc4 (SEQ IDNO:4), or fragments thereof. Another embodiment is directed to anisolated polypeptide NtMyc4 and having at least 85%, 90%, 95%, 96%, 97%,98%, and 99% sequence identity to SEQ ID NO:4, or variant fragmentsthereof.

Another embodiment is directed to an expression vector comprising anisolated cDNA encoding NtMyc3 (SEQ ID NO:1), or fragments thereof.Another embodiment is directed to an expression vector comprising anisolated cDNA encoding NtMyc3 and having at least 85%, 90%, 95%, 96%,97%, 98%, and 99% sequence identity to SEQ ID NO:1, or fragmentsthereof.

Another embodiment is directed to an expression vector comprising anisolated cDNA encoding NtMyc4 (SEQ ID NO:3), or fragments thereof.Another embodiment is directed to an expression vector comprising anisolated cDNA encoding NtMyc4 and having at least 85%, 90%, 95%, 96%,97%, 98%, and 99% sequence identity to SEQ ID NO:3, or fragmentsthereof.

Another embodiment is directed to an expression vector comprising anisolated cDNA encoding NtMyc3 and having at least 85%, 90%, 95%, 96%,97%, 98%, and 99% sequence identity to SEQ ID NO:1, or fragmentsthereof; and an isolated cDNA encoding NtMyc4 and having at least 85%,90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3, orfragments thereof.

Another embodiment is directed to an expression vector comprising cDNAencoding a Myc transcriptional factor and/or cDNA encoding a ERFtranscriptional factor. In particular, this embodiment is directed to anexpression vector comprising: a first sequence comprising an isolatedcDNA encoding NtMyc3 and having at least 85%, 90%, 95%, 96%, 97%, 98%,and 99% sequence identity to SEQ ID NO:1, or fragments thereof; and/or asecond sequence comprising an isolated cDNA encoding NtMyc4 and havingat least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQID NO:3, or fragments thereof; and an isolated cDNA encoding NtERF2 andhaving at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identityto SEQ ID NO:5, or fragments thereof; and/or an isolated cDNA encodingNtERF5 and having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99%sequence identity to SEQ ID NO:6, or fragments thereof, wherein the SEQID NO:5 represents the cDNA sequence encoding NtERF2, and the SEQ IDNO:6 represents the cDNA sequence encoding NtERF5.

Another embodiment is directed to a plant cell line comprising anexpression vector comprising an isolated cDNA encoding NtMyc3 and havingat least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQID NO:1, or fragments thereof; and/or an isolated cDNA encoding NtMyc4and having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequenceidentity to SEQ ID NO:3, or fragments thereof.

Another embodiment is directed to a plant cell line comprising anexpression vector comprising at least one ERF transcriptional factor andat least one Myc transcriptional factor. In particular, this embodimentis directed to a plant cell line comprising an expression vectorcomprising an isolated cDNA encoding NtMyc3 and having at least 85%,90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:1, orfragments thereof; and/or an isolated cDNA encoding NtMyc4 and having atleast 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ IDNO:3, or fragments thereof; and an isolated cDNA encoding NtERF2 andhaving at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identityto SEQ ID NO:5, or fragments thereof; and/or an isolated cDNA encodingNtERF5 and having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99%sequence identity to SEQ ID NO:6, or fragments thereof, wherein the SEQID NO:5 represents the cDNA sequence encoding NtERF2, and the SEQ IDNO:6 represents the cDNA sequence encoding NtERF5.

Another embodiment is directed to a transgenic plant comprising anexpression vector comprising an isolated cDNA encoding NtMyc3 and havingat least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQID NO:1, or fragments thereof; and/or an isolated cDNA encoding NtMyc4and having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequenceidentity to SEQ ID NO:3, or fragments thereof. In a preferredembodiment, the transgenic plant is a tobacco plant.

Another embodiment is directed to a transgenic plant comprising anexpression vector comprising at least one Myc transcriptional factor andat least one ERF transcriptional factor. Another embodiment is directedto a transgenic plant comprising an expression vector comprising anisolated cDNA encoding NtMyc3 and having at least 85%, 90%, 95%, 96%,97%, 98%, and 99% sequence identity to SEQ ID NO:1, or fragmentsthereof; and/or an isolated cDNA encoding NtMyc4 and having at least85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3,or fragments thereof; and an isolated cDNA encoding NtERF2 and having atleast 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ IDNO:5, or fragments thereof; and/or an isolated cDNA encoding NtERF5 andhaving at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identityto SEQ ID NO:6, or fragments thereof, wherein the SEQ ID NO:5 representsthe cDNA sequence encoding NtERF2, and the SEQ ID NO:6 represents thecDNA sequence encoding NtERF5. In a preferred embodiment, the transgenicplant is a tobacco plant.

Another embodiment is directed to a method for genetically regulatingnicotine levels in plants, comprising introducing into a plant anexpression vector comprising an isolated cDNA encoding NtMyc3 and havingat least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQID NO:1, or fragments thereof; and/or an isolated cDNA encoding NtMyc4and having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequenceidentity to SEQ ID NO:3, or fragments thereof. In a preferredembodiment, the transgenic plant is a tobacco plant.

Another embodiment is directed to a method for genetically regulatingnicotine levels in plants, comprising introducing into a plant anexpression vector comprising an isolated cDNA encoding NtMyc3 and havingat least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQID NO:1, or fragments thereof; and/or an isolated cDNA encoding NtMyc4and having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequenceidentity to SEQ ID NO:3, or fragments thereof; and an isolated cDNAencoding NtERF2 and having at least 85%, 90%, 95%, 96%, 97%, 98%, and99% sequence identity to SEQ ID NO:5, or fragments thereof; and/or anisolated cDNA encoding NtERF5 and having at least 85%, 90%, 95%, 96%,97%, 98%, and 99% sequence identity to SEQ ID NO:6, or fragmentsthereof, wherein the SEQ ID NO:5 represents the cDNA sequence encodingNtERF2, and the SEQ ID NO:6 represents the cDNA sequence encodingNtERF5. In a preferred embodiment, the transgenic plant is a tobaccoplant. In a preferred embodiment, the nicotine level in thegenetically-modified plant is increased. In another preferredembodiment, the nicotine level in the genetically-modified plant isdecreased.

Various embodiments are directed to seeds derived fromgenetically-modified transgenic plants described herein.

Various embodiments are directed to various polynucleotide moleculesthat can suppress the expression levels of genes involved in thebiosynthetic pathways for the production of alkaloids, flavonoids, andnicotine. Examples of suitable compositions include ERF and Mycanti-sense polynucleotides that are complementary to ERF and Myctranscript sequences, such as RNAi molecules, microRNAs, and otherdominant negative constructs known to persons skilled in the art.

It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

We claim:
 1. An isolated cDNA molecule comprising a nucleotide sequencehaving at least 95% sequence identity to the nucleotide sequence of SEQID NO:1.
 2. A recombinant polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence encodedby the nucleotide sequence of SEQ ID NO:1.
 3. An expression vectorcomprising the cDNA molecule of claim
 1. 4. A tobacco plant cellcomprising the expression vector of claim
 3. 5. A tobacco plantcomprising the tobacco plant cell of claim 4.